Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification.

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kit) cells. The adult heart indeed contains a heterogeneous mixture of c-kit cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kit cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kit cardiac cells were separated through CD45-positive or -negative sorting followed by c-kit sorting. The blood/endothelial lineage-committed (Lineage) CD45c-kit cardiac cells were compared to CD45(Lineage/Lin) c-kit cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kit cardiac cells are blood/endothelial lineage-committed CD45CD31c-kit cells. In contrast, the LinCD45c-kit cardiac cell cohort, which represents ⩽10% of the total c-kit cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kit and the blood/endothelial lineage-committed c-kit cardiac cells. Single Linc-kit cell-derived clones, which represent only 1-2% of total c-kit myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linc-kit cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kit cardiac cells were injected. Thus, among the cardiac c-kit cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.